Project Summary Microglia are strongly implicated in the pathogenesis of Alzheimer's disease (AD) including late onset sporadic forms of the disease (LOAD). In addition to genetic studies that have identified microglial-enriched genetic variants that influence AD risk, recent computational analysis of multi-scale omics data from hundreds of human LOAD postmortem brains from our group and others in the NIA AMP-AD consortium suggest about one third of the genes associated with risk are enriched or exclusively expressed in microglia. While neurons may be the major cell type generating the toxic amyloid-beta peptide, the functional role of microglia in AD and their interaction with other cell types in the brain to cause disease are still poorly understood and the etiology of AD remains elusive. Key genetic variants in TYROBP/DAP12, TREM2, and APOE may have a functional disease altering impact in distinct brain cell type or interact across cell types in the brain. In this application, we propose to systematically identify and characterize the response of microglia to AD-associated insults in the context of these variants. To study the role of these genes and their functional interaction in AD, we will first generate a panel of CRISPR/Cas9-edited iPSC lines with isogenic mutations in TYROBP/DAP12, TREM2, and APOE in all single and multi-allelic combinations and in the context of a single genetic background with clinical and pathology confirmed LOAD. We will then generate hiPSC-derived neural co-culture systems and then complex organoids from these isogenic lines to characterize the transcriptional and functional impact of key genetic variants in single cell and cell-population-wide analyses. Single cell RNA sequencing data will be generated to identify perturbation signatures for multi-allelic variants that will then be mapped to subtype specific networks to build comprehensive signaling maps for each variant. Functional assays will be used to build evidence for relevance to AD phenotypes. Our overall goal is to test the hypothesis that genetic variants in TREM2, TYROBP/DAP12, and APOE will produce changes in iPSC-derived microglia that mimic the response of microglia to AD-associated insults.